We are studying the control of the human epsilon-globin gene to help understand hemoglobin switching. A previously identified silencing motif in the 5' region of the human epsilon-globin gene when deleted or mutated allows epsilon-globin gene expression in adult cells; other 5' regions also appear to be silencers. These regions in humans show homology with the mouse beta-globin gene cluster. Our experiments involve the use of gene targeting and the Cre-lox site specific recombination system to create "knock-outs" in mice for the functional dissection of these complex DNA regulatory elements in the 5' flanking regions of the mouse epsilon -y-globin gene. Knock-out experiments illustrate more precision in terms of studying the targeted deletion of a gene at its original location verses transgenic experiments, where the location of the gene is not known and multiple copies can be inserted. This approach should define better the physiological importance of DNA regions that were previously identified in our laboratory in cellular assays. In addition to these studies on transcriptional control, we are interested in RNA splicing which may play a secondary role in regulating globin gene expression, particularly in specific mutations leading to beta-thalassemia. This research program involves the investigation of the effect of hemin on beta-thalassemia mRNA splicing in two HeLa cell lines, which were stably transfected with a beta-globin gene that possessed the IVS-II-654(C-T) beta-globin mutation. The HeLa cell lines were treated with varying concentrations of hemin and the effect of hemin was tested by means of RT-PCR, Southern hybridization and restriction endonuclease digestion methods. Recently, erythroid K562 cells, have been stably transfected with beta-globin genes that also possessed IVS-II-654(C-T) beta-globin mutation and experiments involving treatment with varying concentrations of hemin are also being pursued to test the mechanism of action of hemin on beta-globin mRNA splicing in an erythroid specific cell environment. Our goal is to investigate whether hemin treatment or exposure to other agents would be of any therapeutic use for the genetic diseases of hemoglobin such as beta-thalassemia.